Structure of the MED7/MED21 heterodimer and reconstitution of a recombinant Mediator middle module complex

Structure of the MED7/MED21 heterodimer and reconstitution of a recombinant Mediator middle module complex

The Mediator of transcriptional regulation is the central coactivator that enables a response of RNA polymerase II (Pol II) to activators and repressors. Yeast Mediator has a size of more than one MDa and consists of 25 different polypeptides. Biochemical studies defined three Mediator modules in yeast, the head (MED17) the middle (MED9/MED10) and the tail (MED15) modules. During this work, an E.coli coexpression-copurification system was developed, which allowed to study pairwise interactions of Mediator middle module subunits. With the help of this system I reconstituted a complex of two essential and conserved yeast Mediator middle module proteins, the MED7/MED21 heterodimer, and solved its crystal structure. The heterodimer forms an extended structure, which spans one third of the Mediator length, and almost the diameter of Pol II. It shows a four helix bundle and a coiled-coil protrusion connected by a flexible hinge. Multiple conserved patches can be identified on the surface, which allow for assembly of the middle module. A combination of the coexpression-copurification system and assembly of subcomplexes allowed the reconstitution of a five-subunit Mediator middle module subcomplex. The reconstituted subcomplex is able to bind Pol II in vitro. MED6 associates with the middle module and forms a bridge to the head module. The potential flexibility of this bridge and the MED7/MED21 hinge can account for changes in Mediator structure upon its binding to Pol II or to activators.

Abstract

The Mediator of transcriptional regulation is the central coactivator that enables a response of RNA polymerase II (Pol II) to activators and repressors. Yeast Mediator has a size of more than one MDa and consists of 25 different polypeptides. Biochemical studies defined three Mediator modules in yeast, the head (MED17) the middle (MED9/MED10) and the tail (MED15) modules. During this work, an E.coli coexpression-copurification system was developed, which allowed to study pairwise interactions of Mediator middle module subunits. With the help of this system I reconstituted a complex of two essential and conserved yeast Mediator middle module proteins, the MED7/MED21 heterodimer, and solved its crystal structure. The heterodimer forms an extended structure, which spans one third of the Mediator length, and almost the diameter of Pol II. It shows a four helix bundle and a coiled-coil protrusion connected by a flexible hinge. Multiple conserved patches can be identified on the surface, which allow for assembly of the middle module. A combination of the coexpression-copurification system and assembly of subcomplexes allowed the reconstitution of a five-subunit Mediator middle module subcomplex. The reconstituted subcomplex is able to bind Pol II in vitro. MED6 associates with the middle module and forms a bridge to the head module. The potential flexibility of this bridge and the MED7/MED21 hinge can account for changes in Mediator structure upon its binding to Pol II or to activators.